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Fault diagnosis is a central aspect of network fault management. Since faults are unavoidable in communication systems, their quick detection and isolation is essential for the robustness, reliability, and accessibility of a system. Fault localization is done using probing, monitoring, with agents, agent less, through WMI etc. Probing technique for fault localization involves placement of probe stations (Probe stations are specially instrumented nodes from where probes can be sent to monitor the network) which affects the diagnosis capability of the probes sent by the probe stations. Probe station locations affect probing efficiency, monitoring capability, and deployment cost. At the same time, probe station selection affects the selection of probe set for fault localization. We are presenting a novel approach for minimal probe station selection with an objective to reduce probe set size that will be used for fault localization. The probe station selected through this approach results into reduced deployment cost as well as it helps in producing smaller probe set for fault localization reducing overall operation cost. We provide experimental evaluation of the proposed algorithm and provided analysis of same with Shadow Node Reduction (SNR) algorithm through simulation results.

Human cytidine deaminases APOBEC3F (A3F) and APOBEC3G (A3G) are host factors that incorporate into virions and restrict virus replication. We labeled HIV-1 particles with yellow fluorescent protein (YFP)-tagged APOBEC3 proteins and examined their association with preintegration complexes (PICs) in infected cells. Labeling of PICs with A3F-YFP, and to a lesser extent A3G-YFP, could be used to visualize PICs in the nuclei, which was dependent on nuclear pore protein Nup153 but not TNPO3. We show that reverse transcription is not required for nuclear import of PICs, indicating that a viral core uncoating event associated with reverse transcription, and the central DNA flap that forms during reverse transcription, are not required for nuclear import. We also quantify association of cytoplasmic PICs with nuclear envelope (NE) and report that capsid mutations that increase or decrease core stability dramatically reduce NE association and nuclear import of PICs. In addition, we find that nuclear PICs remain close to the NE and are not distributed throughout the nuclei. These results provide tools for tracking retroviral PICs in infected cells and reveal insights into HIV-1 replication.

Several members of the APOBEC3 family of cellular restriction factors provide intrinsic immunity to the host against viral infection. Specifically, APOBEC3DE, APOBEC3F, APOBEC3G, and APOBEC3H haplotypes II, V, and VII provide protection against HIV-1Δvif through hypermutation of the viral genome, inhibition of reverse transcription, and inhibition of viral DNA integration into the host genome. HIV-1 counteracts APOBEC3 proteins by encoding the viral protein Vif, which contains distinct domains that specifically interact with these APOBEC3 proteins to ensure their proteasomal degradation, allowing virus replication to proceed. Here, we review our current understanding of APOBEC3 structure, editing and non-editing mechanisms of APOBEC3-mediated restriction, Vif-APOBEC3 interactions that trigger APOBEC3 degradation, and the contribution of APOBEC3 proteins to restriction and control of HIV-1 replication in infected patients.

Minus-strand DNA transfer, an essential step in retroviral reverse transcription, is mediated by the two repeat (R) regions in the viral genome. It is unclear whether R simply serves as a homologous sequence to mediate the strand transfer or contains specific sequences to promote strand transfer. To test the hypothesis that the molecular mechanism by which R mediates strand transfer is based on homology rather than specific sequences, we examined whether nonviral sequences can be used to facilitate minus-strand DNA transfer. The green fluorescent protein (GFP) gene was divided into GF and FP fragments, containing the 5′ and 3′ portions of GFP, respectively, with an overlapping F fragment (85 bp). FP and GF were inserted into the 5′ and 3′ long terminal repeats, respectively, of a murine leukemia virus-based vector. Utilization of the F fragment to mediate minus-strand DNA transfer should reconstitute GFP during reverse transcription. Flow cytometry analyses demonstrated that GFP was expressed in 73 to 92% of the infected cells, depending on the structure of the viral construct. This indicated that GFP was reconstituted at a high frequency; molecular characterization further confirmed the accurate reconstitution of GFP. These data indicated that nonviral sequences could be used to efficiently mediate minus-strand DNA transfer. Therefore, placement and homology, not specific sequence context, are the important elements in R for minus-strand DNA transfer. In addition, these experiments demonstrate that minus-strand DNA transfer can be used to efficiently reconstitute genes for gene therapy applications.

The antiretroviral nucleoside analog 2′,3′-dideoxy-3′-thiacytidine (3TC) is a potent inhibitor of wild-type human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). A methionine-to-valine or methionine-to-isoleucine substitution at residue 184 in the HIV-1 YMDD motif, which is located at the RT active site, leads to a high level of resistance to 3TC. We sought to determine whether 3TC can inhibit the replication of wild-type murine leukemia virus (MLV), which contains V223 at the YVDD active site motif of the MLV RT, and of the V223M, V223I, V223A, and V223S mutant RTs. Surprisingly, the wild type and all four of the V223 mutants of MLV RT were highly resistant to 3TC. These results indicate that determinants outside the YVDD motif of MLV RT confer a high level of resistance to 3TC. Therefore, structural differences among similar RTs might result in widely divergent sensitivities to antiretroviral nucleoside analogs.

Error-prone DNA synthesis by retroviral reverse transcriptases (RTs) is a major contributor to variation in retroviral populations. Structural features of retroviral RTs that are important for accuracy of DNA synthesis in vivo are not known. To identify structural elements of murine leukemia virus (MLV) RT important for fidelity in vivo, we developed a D17-based encapsidating cell line (ANGIE P) which is designed to express the amphotropic MLV envelope. ANGIE P also contains an MLV-based retroviral vector (GA-1) which encodes a wild-type bacterial β-galactosidase gene ( lacZ ) and a neomycin phosphotransferase gene. Transfection of ANGIE P cells with wild-type or mutated MLV gag-pol expression constructs generated GA-1 virus that was able to undergo only one cycle of viral replication upon infection of D17 cells. The infected D17 cell clones were characterized by staining with 5-bromo-4-chloro-3-indolyl-β- d -galactopyranoside (X-Gal), and the frequencies of inactivating mutations in lacZ were quantified. Three mutations in the YVDD motif (V223M, V223S, and V223A) and two mutations in the RNase H domain (S526A and R657S) exhibited frequencies of lacZ inactivation 1.2- to 2.3-fold higher than that for the wild-type MLV RT ( P < 0.005). Two mutations (V223I and Y598V) did not affect the frequency of lacZ inactivation. These results establish a sensitive in vivo assay for identification of structural determinants important for accuracy of DNA synthesis and indicate that several structural determinants may have an effect on the in vivo fidelity of MLV RT.

Deletion of direct repeats in retroviral genomes provides an in vivo system for analysis of reverse transcriptase (RT) template switching. The effect of distance between direct repeats on the rate of deletion was determined for 16 murine leukemia virus (MLV)-based vectors containing a 701-bp direct repeat of overlapping fragments of the herpes simplex virus thymidine kinase gene (HTK). The direct repeats were separated by spacer fragments of various lengths (0.1 to 3.5 kb). Southern analysis of infected cells after one replication cycle indicated that all vectors in which the distance between homologous sequences was >1,500 bp deleted at very high rates (>90%). In contrast, vectors containing

Expression of the selectable drug resistance gene in retroviral vectors used for gene therapy can lead to a decreased expression of the gene of interest and may induce a host immune response, resulting in a decreased efficiency of gene therapy. In this study, we demonstrate that high-frequency deletion of direct repeats, an inherent property of reverse transcriptases, can be used to efficiently excise the drug resistance gene during reverse transcription. One retroviral vector containing a direct repeat deleted the neomycin resistance expression cassette during a single replication cycle at >99% efficiency.

Deoxyribonucleoside triphosphate (dNTP) pool imbalances are associated with an increase in the rate of misincorporation and hypermutation during in vitro reverse transcription reactions. However, the effects of in vivo dNTP pool imbalances on the accuracy of reverse transcription are unknown. We sought to determine the effects of in vivo dNTP pool imbalances on retroviral mutation rates and to test our hypothesis that 3′-azido-3′-deoxythymidine (AZT) increases the retroviral mutation rates through induction of dNTP pool imbalances. D17 cells were treated with thymidine, hydroxyurea (HU), or AZT, and the effects on in vivo dNTP pools were measured. Thymidine and HU treatments induced significant dNTP pool imbalances. In contrast, AZT treatment had very little effect on the dNTP pools. The effects of in vivo dNTP pool imbalances induced by thymidine and HU treatments on the retroviral mutation rates were also determined. Spleen necrosis virus (SNV)-based and murine leukemia virus (MLV)-based retroviral vectors that expressed the lacZ mutant reporter gene were used. The frequencies of inactivating mutations introduced in the lacZ gene in a single replication cycle provided a measure of the retroviral mutation rates. Treatment of D17 target cells with 500 μM thymidine increased the SNV and MLV mutant frequencies 4.7- and 4-fold, respectively. Treatment of D17 target cells with 2 mM HU increased the SNV and MLV mutant frequencies 2.1- and 2.7-fold, respectively. These results demonstrate that dNTP pool imbalances are associated with an increase in the in vivo retroviral mutation rates, but AZT treatment results in an increase in the retroviral mutation rates by a mechanism not involving alterations in dNTP pools.

Retroviral reverse transcriptases (RTs) frequently switch templates during DNA synthesis, which can result in mutations and recombination. The relative rates of in vivo RT template switching during RNA- and DNA-dependent DNA synthesis are unknown. To determine the relative rates of RT template switching during copying of RNA and DNA templates, we constructed spleen necrosis virus-based retroviral vectors containing a 400-bp direct repeat. The directly repeated sequences were upstream of the polypurine tract (PPT) in the RB-LLP vector; the same direct repeats flanked the PPT and attachment site ( att ) in the RB-LPL vector. RT template switching events could occur during either RNA- or DNA-dependent DNA synthesis and delete one copy of the direct repeat plus the intervening sequences. RB-LLP vectors that underwent direct repeat deletions during RNA- and DNA-dependent DNA synthesis generated viral DNA that could integrate into the host genome. However, any deletion of the direct repeats in the RB-LPL vector that occurred during RNA-dependent DNA synthesis resulted in deletion of the essential PPT and att site and generated a dead-end viral DNA product. Thus, only RB-LPL vectors that underwent direct repeat deletions during DNA-dependent DNA synthesis could integrate to form proviruses. The RB-LLP and RB-LPL vectors were permitted to undergo a single replication cycle, and the frequencies of direct repeat deletions were determined by PCR and Southern analysis of the resulting proviruses. A comparison of the frequency of direct repeat deletions in the RB-LLP and RB-LPL vectors indicated that the in vivo rates of RT template switching during RNA- and DNA-dependent DNA synthesis are nearly identical.

Homologous recombination and deletions occur during retroviral replication when reverse transcriptase switches templates. While recombination occurs solely by intermolecular template switching (between copackaged RNAs), deletions can occur by an intermolecular or an intramolecular template switch (within the same RNA). To directly compare the rates of intramolecular and intermolecular template switching, two spleen necrosis virus-based vectors were constructed. Each vector contained a 110-bp direct repeat that was previously shown to delete at a high rate. The 110-bp direct repeat was flanked by two different sets of restriction site markers. These vectors were used to form heterozygotic virions containing RNAs of each parental vector, from which recombinant viruses were generated. By analyses of the markers flanking the direct repeats in recombinant and nonrecombinant proviruses, the rates of intramolecular and intermolecular template switching were determined. The results of these analyses indicate that intramolecular template switching is much more efficient than intermolecular template switching and that direct repeat deletions occur primarily through intramolecular template switching events. These studies also indicate that retroviral recombination occurs within a distinct viral subpopulation and exhibits high negative interference, whereby the selection of one recombination event increases the probability that a second recombination event will be observed.

It was previously observed that the nucleoside analog 5-azacytidine increased the spleen necrosis virus (SNV) mutation rate 13-fold in one cycle of retrovirus replication (V. K. Pathak and H. M. Temin, J. Virol. 66:3093-3100, 1992). Based on this observation, we hypothesized that nucleoside analogs used as antiviral drugs may also increase retrovirus mutation rates. We sought to determine if 3'-azido-3'-deoxythymidine (AZT), the primary treatment for human immunodeficiency virus type 1 (HIV-1) infection, increases the retrovirus mutation rate. Two assays were used to determine the effects of AZT on retrovirus mutation rates. The strategy of the first assay involved measuring the in vivo rate of inactivation of the lacZ gene in one replication cycle of SNV- and murine leukemia virus-based retroviral vectors. We observed 7- and 10-fold increases in the SNV mutant frequency following treatment of target cells with 0.1 and 0.5 microM AZT, respectively. The murine leukemia virus mutant frequency increased two- and threefold following treatment of target cells with 0.5 and 1.0 microM AZT, respectively. The second assay used an SNV-based shuttle vector containing the lacZ alpha gene. Proviruses were recovered as plasmids in Escherichia coli, and the rate of inactivation of lacZ alpha was measured. The results indicated that treatment of target cells increased the overall mutation rate two- to threefold. DNA sequence analysis of mutant proviruses indicated that AZT increased both the deletion and substitution rates. These results suggest that AZT treatment of HIV-1 infection may increase the degree of viral variation and alter virus evolution or pathogenesis.

During reverse transcription, minus-strand DNA transfer connects the sequences located at the two ends of the viral RNA to generate a long terminal repeat. It is thought that the homology in the repeat (R) regions located at the two ends of the viral RNA sequences facilitate minus-strand DNA transfer. In this report, the effects of diminished R-region homology on DNA synthesis and virus titer were examined. A retrovirus vector, PY31, was constructed to contain the 5' and 3' cis-acting elements from Moloney murine sarcoma virus and spleen necrosis virus. These two viruses are genetically distinct, and the two R regions contain little homology. In one round of replication, the PY31 titer was approximately 3,000-fold lower than that of a control vector with highly homologous R regions. The molecular characteristics of the junctions of minus-strand DNA transfer were analyzed in both unintegrated DNA and integrated proviruses. Short stretches of homology were found at the transfer junctions and were likely to be used to facilitate minus-strand DNA transfer. Both minus-strand strong-stop DNA and weak-stop DNA were observed to mediate strand transfer. The ability of PY31 to complete reverse transcription indicates that minus-strand DNA transfer can be used to join sequences from two different viruses to form recombinant viruses. These results suggest the provocative possibility that genetically distinct viruses can interact through this mechanism.

High evolutionary potential and genetic variation in retroviral populations depend upon the mutation and recombination rates per replication cycle, the replication rate (replication cycles per time), and the selective forces that act on the population. The virally encoded reverse transcriptases lack proofreading capability and are evolutionarily selected for jumping (switching templates) during reverse transcription. One consequence of the template switching nature of reverse transcriptases is a high rate of intramolecular template switching, which leads to defective genome formation; another consequence is intermolecular template switching between two copackaged genomic RNAs, which leads to recombination.

This paper is devoted largely for building a novel approach for human recognition using eye movement analysis. Velocity and dispersion threshold based fixation identification algorithms are employed for processing the raw scan path signals in oculo-motion matrices. A new hybrid intelligent model is deployed for classification over data retrieved from scan-path signals. Experimental results demonstrate the endeavor of oculo-motion signals as an effective biometric trait. This paper also demonstrates the relative comparison of the two fixation identification techniques combined with hybrid intelligent model.

Background Xenotropic Murine leukemia virus-Related Virus (XMRV) is a γ-retrovirus initially reported to be present within familial human prostate tumors and the blood of patients with chronic fatigue syndrome. Subsequent studies however were unable to replicate these findings, and there is now compelling evidence that the virus evolved through rare retroviral recombination events in human tumor cell lines established through murine xenograft experiments. There is also no direct evidence that XMRV infection has any functional effects that contribute to tumor pathogenesis. Results Herein we describe an additional xenotropic MLV, “B4rv”, found in a cell line derived from xenograft experiments with the human prostate cancer LNCaP cell line. When injected subcutaneously in nude mice, LNCaP cells infected with XMRV or B4rv formed larger tumors that were highly hemorrhagic and displayed poor pericyte/smooth muscle cell (SMC) investment, markers of increased metastatic potential. Conditioned media derived from XMRV- or B4rv-infected LNCaPs, but not an amphotropic MLV control virus infected LNCaPs, profoundly decreased expression of marker genes in cultured SMC, consistent with inhibition of SMC differentiation/maturation. Similar effects were seen with a chimeric virus of the amphotropic MLV control virus containing the XMRV env gene, but not with an XMRV chimeric virus containing the amphotropic MLV env gene. UV-inactivated XMRV and pseudovirions that were pseudotyped with XMRV envelope protein also produce conditioned media that down-regulated SMC marker gene expression in vitro. Conclusions Together these results indicate that xenotropic MLV envelope proteins are sufficient to induce the production of factors by tumor cells that suppress vascular SMC differentiation, providing evidence for a novel mechanism by which xenotropic MLVs might alter tumor pathogenesis by disrupting tumor vascular maturation. Although it is highly unlikely that either XMRV or B4Rv themselves infect humans and are pathogenic, the results suggest that xenograft approaches commonly used in the study of human cancer promote the evolution of novel retroviruses with pathogenic properties.

Fault diagnosis is a central aspect of network fault management. Since faults are unavoidable in communication systems, their quick detection and isolation is essential for the robustness, reliability, and accessibility of a system. Probing technique for fault localization involves placement of probe stations (Probe stations are specially instrumented nodes from where probes can be sent to monitor the network) which affects the diagnosis capability of the probes sent by the probe stations. Probe station locations affect probing efficiency, monitoring capability, and deployment cost. At the same time, probe station selection affects the selection of probe set for fault localization. We are presenting a novel approach for minimal probe station selection with an objective to reduce probe set size that will be used for fault localization. The probe station selected through this approach results into reduced deployment cost as well as it helps in producing smaller probe set for fault localization reducing overall operation cost. We provide experimental evaluation of the proposed algorithms through simulation results.

We have developed novel self-inactivating and self-activating retroviral vectors based on the previously observed high-frequency deletion of direct repeats. We constructed spleen necrosis virus (SNV)-based viral vectors that contained large direct repeats flanking the viral encapsidation sequence (E). A large proportion of the proviruses in the target cells had E and one copy of the direct repeat deleted. Direct repeats of 1,333 and 788 bp were deleted at frequencies of 93 and 85%, respectively. To achieve a 100% deletion efficiency in target cells after ex vivo infection and drug selection, we constructed a self-activating vector that simultaneously deleted E and reconstituted the neomycin phosphotransferase gene. Selection of the target cells for resistance to G418 (a neomycin analog) ensured that all integrated proviruses had E deleted. The proviruses with E deleted were mobilized by a replication-competent virus 267,000-fold less efficiently than proviruses with E. We named these self-inactivating vectors E- (E-minus) vectors. These vectors should increase the safety of retroviral vector-mediated gene therapy by preventing the spread of vector sequences to nontarget cells in the event of coinfection with helper virus. We propose that direct-repeat deletions occur during RNA-dependent DNA synthesis and suggest that template switches occur without a requirement for RNA breaks. The minimum template dissociation frequency was estimated as 8%/100 bp per replication cycle. These vectors demonstrate that large direct repeats and template-switching properties of reverse transcriptase can be utilized to delete any sequence or reconstitute genes during retroviral replication.

APOBEC3 proteins inhibit HIV-1 replication in experimental systems and induce hypermutation in infected patients; however, the relative contributions of several APOBEC3 proteins to restriction of HIV-1 replication in the absence of the viral Vif protein in human primary CD4 + T cells and macrophages are unknown. We observed significant inhibition of HIV-1Δ vif produced in 293T cells in the presence of APOBEC3DE (A3DE), APOBEC3F (A3F), APOBEC3G (A3G), and APOBEC3H haplotype II (A3H HapII) but not APOBEC3B (A3B), APOBEC3C (A3C), or APOBEC3H haplotype I (A3H HapI). Our previous studies showed that Vif amino acids Y 40 RHHY 44 are important for inducing proteasomal degradation of A3G, whereas amino acids 14 DRMR 17 are important for degradation of A3F and A3DE. Here, we introduced substitution mutations of 40 YRHHY 44 and 14 DRMR 17 in replication-competent HIV-1 to generate vif mutants NL4-3 YRHHY>A5 and NL4-3 DRMR>A4 to compare the antiviral activity of A3G to the combined antiviral activity of A3F and A3DE in activated CD4 + T cells and macrophages. During the first 15 days (round 1), in which multiple cycles of viral replication occurred, both the NL4-3 YRHHY>A5 and NL4-3 DRMR>A4 mutants replicated in activated CD4 + T cells and macrophages, and only the NL4-3 YRHHY>A5 mutant showed a 2- to 4-day delay in replication compared to the wild type. During the subsequent 27 days (round 2) of cultures initiated with peak virus obtained from round 1, the NL4-3 YRHHY>A5 mutant exhibited a longer, 8- to 10-day delay and the NL4-3 DRMR>A4 mutant exhibited a 2- to 6-day delay in replication compared to the wild type. The NL4-3 YRHHY>A5 and NL4-3 DRMR>A4 mutant proviruses displayed G-to-A hypermutations primarily in GG and GA dinucleotides as expected of A3G- and A3F- or A3DE-mediated deamination, respectively. We conclude that A3G exerts a greater restriction effect on HIV-1 than A3F and A3DE.

Mutations in the connection subdomain (CN) and RNase H domain (RH) of HIV-1 reverse transcriptase (RT) from subtype B-infected patients enhance nucleoside and nonnucleoside RT inhibitor (NRTI and NNRTI) resistance by affecting the balance between polymerization and RNase H activity. To determine whether CN mutations in subtype C influence drug sensitivity, single genome sequencing was performed on Brazilian subtype C-infected patients failing RTI therapy. CN mutations identified were similar to subtype B, including A376S, A400T, Q334D, G335D, N348I, and A371V, and increased AZT resistance in the presence of thymidine analog mutations. CN mutations also enhanced NNRTI resistance in the presence of classical NNRTI mutations: etravirine resistance was enhanced 6- to 11-fold in the presence of L100I/K103N/Y181C. These results indicate that selection of CN mutations in treatment-experienced patients also occurs in subtype-C-infected patients and are likely to provide valuable information in predicting clinical RTI resistance.

The discovery and de-discovery of the xenotropic murine leukemia virus-related virus (XMRV) has been a tumultuous roller-coaster ride for scientists and patients. The initial associations of XMRV with chronic fatigue syndrome and prostate cancer, while providing much hope and optimism, have now been discredited and/or retracted following overwhelming evidence that (1) numerous patient cohorts from around the world are XMRV-negative, (2) the initial reports of XMRV-positive patients were due to contamination with mouse DNA, XMRV plasmid DNA, or virus from the 22Rv1 cell line and (3) XMRV is a laboratory-derived virus generated in the mid 1990s through recombination during passage of a prostate tumor xenograft in immuno-compromised mice. While these developments are disappointing to scientists and patients, they provide a valuable road map of potential pitfalls to the would-be microbe hunters.

Although xenotropic murine leukemia virus-related virus (XMRV) has been previously linked to prostate cancer and myalgic encephalomyelitis/chronic fatigue syndrome, recent data indicate that results interpreted as evidence of human XMRV infection reflect laboratory contamination rather than authentic in vivo infection. Nevertheless, XMRV is a retrovirus of undefined pathogenic potential that is able to replicate in human cells. Here we describe a comprehensive analysis of two male pigtailed macaques ( Macaca nemestrina ) experimentally infected with XMRV. Following intravenous inoculation with >10 10 RNA copy equivalents of XMRV, viral replication was limited and transient, peaking at ≤2,200 viral RNA (vRNA) copies/ml plasma and becoming undetectable by 4 weeks postinfection, though viral DNA (vDNA) in peripheral blood mononuclear cells remained detectable through 119 days of follow-up. Similarly, vRNA was not detectable in lymph nodes by in situ hybridization despite detectable vDNA. Sequencing of cell-associated vDNA revealed extensive G-to-A hypermutation, suggestive of APOBEC-mediated viral restriction. Consistent with limited viral replication, we found transient upregulation of type I interferon responses that returned to baseline by 2 weeks postinfection, no detectable cellular immune responses, and limited or no spread to prostate tissue. Antibody responses, including neutralizing antibodies, however, were detectable by 2 weeks postinfection and maintained throughout the study. Both animals were healthy for the duration of follow-up. These findings indicate that XMRV replication and spread were limited in pigtailed macaques, predominantly by APOBEC-mediated hypermutation. Given that human APOBEC proteins restrict XMRV infection in vitro , human XMRV infection, if it occurred, would be expected to be characterized by similarly limited viral replication and spread.

The retrovirus XMRV (xenotropic murine leukemia virus–related virus) has been detected in human prostate tumors and in blood samples from patients with chronic fatigue syndrome, but these findings have not been replicated. We hypothesized that an understanding of when and how XMRV first arose might help explain the discrepant results. We studied human prostate cancer cell lines CWR22R v1 and CWR-R1, which produce XMRV virtually identical to the viruses recently found in patient samples, as well as their progenitor human prostate tumor xenograft (CWR22) that had been passaged in mice. We detected XMRV infection in the two cell lines and in the later passage xenografts, but not in the early passages. In particular, we found that the host mice contained two proviruses, PreXMRV-1 and PreXMRV-2, which share 99.92% identity with XMRV over >3.2-kilobase stretches of their genomes. We conclude that XMRV was not present in the original CWR22 tumor but was generated by recombination of two proviruses during tumor passaging in mice. The probability that an identical recombinant was generated independently is negligible (~10 –12 ); our results suggest that the association of XMRV with human disease is due to contamination of human samples with virus originating from this recombination event.

The MLV-related retrovirus, XMRV, was recently identified and reported to be associated with both prostate cancer and chronic fatigue syndrome. At the National Cancer Institute-Frederick, MD (NCI-Frederick), we developed highly sensitive methods to detect XMRV nucleic acids, antibodies, and replication competent virus. Analysis of XMRV-spiked samples and/or specimens from two pigtail macaques experimentally inoculated with 22Rv1 cell-derived XMRV confirmed the ability of the assays used to detect XMRV RNA and DNA, and culture isolatable virus when present, along with XMRV reactive antibody responses. Using these assays, we did not detect evidence of XMRV in blood samples () or prostate specimens () from two independent cohorts of patients with prostate cancer. Previous studies detected XMRV in prostate tissues. In the present study, we primarily investigated the levels of XMRV in blood plasma samples collected from patients with prostate cancer. These results demonstrate that while XMRV-related assays developed at the NCI-Frederick can readily measure XMRV nucleic acids, antibodies, and replication competent virus, no evidence of XMRV was found in the blood of patients with prostate cancer.

In this paper, we present a novel evolutionary fuzzy clustering approach with Minkowski distances. Fuzzy clustering plays an important role for various kinds of classification problems. Evolutionary algorithm is used for searching the best partitioning among the populations generated by different runs of the fuzzy clustering algorithm. Evolutionary fuzzy clustering performs better as compared to the conventional fuzzy clustering in terms of classification accuracy and partitioning. Fuzzy c-means (FCM) is a data clustering algorithm in which each data point is associated with a cluster through a membership degree. Here, Minkowski distance is used with FCM instead of conventional Euclidian distance because of its more generalized nature. It does not restrict the shape of the clusters generated. Empirical evaluation demonstrates the performance of proposed novel technique in terms of precision and accuracy in various benchmark problems.

This paper proposes a method of geometric modeling of features extracted from 3D face and presents some of its applications. It describes a new automatic pose and expression invariant feature extraction algorithms to extract features (control points) from eyebrows, nose and lips of 3D facial data. The proposed algorithms are tested on BU-3DFE (Binghamton University 3D Facial Expression) Database where each subject has at least six expressions. Three dimensional curves are fitted on these extracted features. Through Chi-Square test it reveals that the curve fitted against each extracted feature is found to be good with 98.5% confidence level. In this paper, the proposed geometric model has been used in 3D face recognition and in regeneration of all features. It has been found that the model helps to reduce the storage space considerably and can be used as a soft biometric tool to classify the biometric images in the database so that search space in the database for identification can be reduced substantially.

High-frequency recombination is a hallmark of HIV-1 replication. Recombination can occur between two members of the same subtype or between viruses from two different subtypes, generating intra- or intersubtype recombinants, respectively. Many intersubtype recombinants have been shown to circulate in human populations. We hypothesize that sequence diversity affects the emergence of viable recombinants by decreasing recombination events and reducing the ability of the recombinants to replicate. To test our hypothesis, we compared recombination between two viruses containing subtype B pol genes (B/B) and between viruses with pol genes from subtype B or F (B/F). Recombination events generated during a single cycle of infection without selection pressure on pol gene function were analyzed by single-genome sequencing. We found that recombination occurred slightly (∼30%) less frequently in B/F than in B/B viruses, and the overall distribution of crossover junctions in pol was similar for the two classes of recombinants. We then examined the emergence of recombinants in a multiple cycle assay, so that functional pol gene products were selected. We found that the emerging B/B recombinants had complex patterns, and the crossover junctions were distributed throughout the pol gene. In contrast, selected B/F recombinants had limited recombination patterns and restricted crossover junction distribution. These results provide evidence for the evolved coadapted sites in variants from different subtypes; these sites may be segregated by recombination events, causing the newly generated intersubtype recombinants to undergo purifying selection. Therefore, the ability of the recombinants to replicate is the major barrier for many of these viruses.

Recently, mutations in the connection subdomain (CN) and RNase H domain of HIV-1 reverse transcriptase (RT) were observed to exhibit dual resistance to nucleoside and nonnucleoside reverse transcriptase inhibitors (NRTIs and NNRTIs). To elucidate the mechanism by which CN and RH mutations confer resistance to NNRTIs, we hypothesized that these mutations reduce RNase H cleavage and provide more time for the NNRTI to dissociate from the RT, resulting in the resumption of DNA synthesis and enhanced NNRTI resistance. We observed that the effect of the reduction in RNase H cleavage on NNRTI resistance is dependent upon the affinity of each NNRTI to the RT and further influenced by the presence of NNRTI-binding pocket (BP) mutants. D549N, Q475A, and Y501A mutants, which reduce RNase H cleavage, enhance resistance to nevirapine (NVP) and delavirdine (DLV), but not to efavirenz (EFV) and etravirine (ETR), consistent with their increase in affinity for RT. Combining the D549N mutant with NNRTI BP mutants further increases NNRTI resistance from 3- to 30-fold, supporting the role of NNRTI-RT affinity in our NNRTI resistance model. We also demonstrated that CNs from treatment-experienced patients, previously reported to enhance NRTI resistance, also reduce RNase H cleavage and enhance NNRTI resistance in the context of the patient RT pol domain or a wild-type pol domain. Together, these results confirm key predictions of our NNRTI resistance model and provide support for a unifying mechanism by which CN and RH mutations can exhibit dual NRTI and NNRTI resistance.

This paper proposes an efficient watermark generation method for audio copyright protection and digital right management. It proposes a method of generating pseudorandom sequences based on iris image templates. These biometric generated sequences (bio-keys) are found to be unique for distinct identification purposes. These bio-keys are used as the watermark for audio signals. The correlation of the extracted bio-key with a database of 150 bio-keys reveals that the best correlation is 1 while the next best correlation is less than 0.4 under no attack condition. Even under serious signal processing attacks, the bio-keys have distinctly been identified and mapped in a database. Experimental results of listening evaluation and robustness tests have confirmed that the use of bio-keys maintains good perceptual property and offers strong resistance to the typical signal processing attacks.

Perceptual transparency and robustness are trade offs in audio watermarking which is an existing research problem. Synchronization attacks are the most challenging attack in audio watermarking. An algorithm is proposed in this paper which has good perceptual transparency and is also robust to synchronization attacks. The watermark embedding regions are selectively chosen in the high energy regions of the audio which makes the embedding process robust to signal processing attacks. The watermark is embedded in the audio in an adaptive manner. The degree of embedding is localized in nature and depends on the audio content of that localized region. The watermark is embedded by quantization of the singular values in the wavelet domain which makes the process perceptually transparent. The experimental results suggest that the proposed method maintains a good perceptual quality of the audio signal and maintains good robustness against signal processing attacks.

This paper proposes a unconventional method for removable audible watermarking system based on the requirements of a promising application. Given a audio file, the system makes some part of file available for preview and perceptual watermarking on the remaining portion. The watermark is embedded into selected DCT coefficients of host audio signal so that the signal to noise ratio is maintained at a level which is audibly annoying to human auditory system. Once the audio file is decoded using the secret key a new watermark is embedded in the audio that is perceptually transparent to the human auditory system. Hence this double watermarking i.e. imperceptible and perceptible watermarking provides a novel prototype for digital right management control. The subjective quality tests and robustness tests indicate that the audio quality is excellent and is robust to signal processing attacks.

This paper proposes an efficient expression invariant algorithm for 3D face recognition of subjects. The proposed algorithm uses a surface-based approach to extract automatically and to define geometrically the facial features like eyebrows, nose and lips. These extracted features are used to obtain some unique control points that can be used for matching. The algorithm is tested on Binghamton University 3D Facial Expression (BU-3DFE) database where each subject is having at least 6 expressions and is found to be more than 98.5% accurate.

The issue of ownership of digital watermark is addressed in this paper. An audio watermarking scheme is presented where the biometric pattern of iris is used to generate the watermark that has a stamp of ownership. The watermark is embedded in the high-energy regions selectively in wavelet domain that makes the embedding process robust against signal processing attacks. The watermark extraction process is blind and does not require the original host audio signal. Experimental results of listening evaluation and robustness tests have confirmed that the proposed scheme maintains good perceptual property and offers strong resistance to typical signal processing attacks.

We previously shown that mutations in the connection (CN) subdomain of human immunodeficiency virus type 1 (HIV-1) subtype B reverse transcriptase (RT) increase 3′-azido-3′-deoxythymidine (AZT) resistance in the context of thymidine analog mutations (TAMs) by affecting the balance between polymerization and RNase H activity. To determine whether this balance affects drug resistance in other HIV-1 subtypes, recombinant subtype CRF01_AE was analyzed. Interestingly, CRF01_AE containing TAMs exhibited 64-fold higher AZT resistance relative to wild-type B, whereas AZT resistance of subtype B containing the same TAMs was 13-fold higher, which in turn correlated with higher levels of AZT-monophosphate (AZTMP) excision on both RNA and DNA templates. The high level of AZT resistance exhibited by CRF01_AE was primarily associated with the T400 residue in wild-type subtype AE CN subdomain. An A400T substitution in subtype B enhanced AZT resistance, increased AZTMP excision on both RNA and DNA templates, and reduced RNase H cleavage. Replacing the T400 residue in CRF01_AE with alanine restored AZT sensitivity and reduced AZTMP excision on both RNA and DNA templates, suggesting that the T400 residue increases AZT resistance in CRF01_AE at least in part by directly increasing the efficiency of AZTMP excision. These results show for the first time that CRF01_AE exhibits higher levels of AZT resistance in the presence of TAMs and that this resistance is primarily associated with T400. Our results also show that mixing the RT polymerase, CN, and RNase H domains from different subtypes can underestimate AZT resistance levels, and they emphasize the need to develop subtype-specific genotypic and phenotypic assays to provide more accurate estimates of clinical drug resistance.

The role of APOBEC3 (A3) protein family members in inhibiting retrovirus infection and mobile element retrotransposition is well established. However, the evolutionary effects these restriction factors may have had on active retroviruses such as HIV-1 are less well understood. An HIV-1 variant that has been highly G-to-A mutated is unlikely to be transmitted due to accumulation of deleterious mutations. However, G-to-A mutated hA3G target sequences within which the mutations are the least deleterious are more likely to survive selection pressure. Thus, among hA3G targets in HIV-1, the ratio of nonsynonymous to synonymous changes will increase with virus generations, leaving a footprint of past activity. To study such footprints in HIV-1 evolution, we developed an in silico model based on calculated hA3G target probabilities derived from G-to-A mutation sequence contexts in the literature. We simulated G-to-A changes iteratively in independent sequential HIV-1 infections until a stop codon was introduced into any gene. In addition to our simulation results, we observed higher ratios of nonsynonymous to synonymous mutation at hA3G targets in extant HIV-1 genomes than in their putative ancestral genomes, compared to random controls, implying that moderate levels of A3G-mediated G-to-A mutation have been a factor in HIV-1 evolution. Results from in vitro passaging experiments of HIV-1 modified to be highly susceptible to hA3G mutagenesis verified our simulation accuracy. We also used our simulation to examine the possible role of A3G-induced mutations in the origin of drug resistance. We found that hA3G activity could have been responsible for only a small increase in mutations at known drug resistance sites and propose that concerns for increased resistance to other antiviral drugs should not prevent Vif from being considered a suitable target for development of new drugs., Author Summary The search for new drugs to battle HIV-1 infections is a continuing struggle. APOBEC3G proteins have been shown to deaminate C-residues in HIV-1 minus strand DNA during its synthesis, resulting in G-to-A mutations in the RNA genome. The HIV-1 Vif protein has evolved to counteract APOBEC3G and thereby escape these frequently deleterious mutations, making Vif an attractive target for new drugs. However, a partial block of Vif could result in an increased although low-level HIV-1 G-to-A mutation rate. Here we investigated APOBEC3G mutation footprints in HIV-1 evolution and the potential risk for known drug resistance from sublethal G-to-A mutations. Using computer simulations, the accuracies of which were verified by infection experiments, we detected evolutionary APOBEC3G mutation footprints in the HIV-1 genome. We predict that the risk that APOBEC3G-induced G-to-A mutations will cause drug resistance is very low. We therefore propose that concerns for increased resistance to other antiviral drugs should not prevent Vif from being considered a suitable target for development of new drugs.

Background Host restriction factor APOBEC3G (A3G) blocks human immunodeficiency virus type 1 (HIV-1) replication by G-to-A hypermutation, and by inhibiting DNA synthesis and provirus formation. Previous reports have suggested that A3G is a dimer and its virion incorporation is mediated through interactions with viral or nonviral RNAs and/or HIV-1 Gag. We have now employed a bimolecular fluorescence complementation assay (BiFC) to analyze the intracellular A3G-A3G, A3G-RNA, and A3G-Gag interactions in living cells by reconstitution of yellow fluorescent protein (YFP) from its N- or C-terminal fragments. Results The results obtained with catalytic domain 1 and 2 (CD1 and CD2) mutants indicate that A3G-A3G and A3G-Gag multimerization is dependent on an intact CD1 domain, which is required for RNA binding. A mutant HIV-1 Gag that exhibits reduced RNA binding also failed to reconstitute BiFC with wild-type A3G, indicating a requirement for both HIV-1 Gag and A3G to bind to RNA for their multimerization. Addition of a non-specific RNA binding peptide (P22) to the N-terminus of a CD1 mutant of A3G restored BiFC and virion incorporation, but failed to inhibit viral replication, indicating that the mutations in CD1 resulted in additional defects that interfere with A3G's antiviral activity. Conclusion These studies establish a robust BiFC assay for analysis of intracellular interactions of A3G with other macromolecules. The results indicate that in vivo A3G is a monomer that forms multimers upon binding to RNA. In addition, we observed weak interactions between wild-type A3G molecules and RNA binding-defective mutants of A3G, which could explain previously described protein-protein interactions between purified A3G molecules.

Background Naturally occurring Vif variants that are unable to inhibit the host restriction factor APOBEC3G (A3G) have been isolated from infected individuals. A3G can potentially induce G-to-A hypermutation in these viruses, and hypermutation could contribute to genetic variation in HIV-1 populations through recombination between hypermutant and wild-type genomes. Thus, hypermutation could contribute to the generation of immune escape and drug resistant variants, but the genetic contribution of hypermutation to the viral evolutionary potential is poorly understood. In addition, the mechanisms by which these viruses persist in the host despite the presence of A3G remain unknown. Results To address these questions, we generated a replication-competent HIV-1 Vif mutant in which the A3G-binding residues of Vif, Y40RHHY44, were substituted with five alanines. As expected, the mutant was severely defective in an A3G-expressing T cell line and exhibited a significant delay in replication kinetics. Analysis of viral DNA showed the expected high level of G-to-A hypermutation; however, we found substantially reduced levels of G-to-A hypermutation in intracellular viral RNA (cRNA), and the levels of G-to-A mutations in virion RNA (vRNA) were even further reduced. The frequencies of hypermutation in DNA, cRNA, and vRNA were 0.73%, 0.12%, and 0.05% of the nucleotides sequenced, indicating a gradient of hypermutation. Additionally, genomes containing start codon mutations and early termination codons within gag were isolated from the vRNA. Conclusion These results suggest that sublethal levels of hypermutation coupled with purifying selection at multiple steps during the early phase of viral replication lead to the packaging of largely unmutated genomes, providing a mechanism by which mutant Vif variants can persist in infected individuals. The persistence of genomes containing mutated gag genes despite this selection pressure indicates that dual infection and complementation can result in the packaging of hypermutated genomes which, through recombination with wild-type genomes, could increase viral genetic variation and contribute to evolution.

Guidelines for Naming Nonprimate APOBEC3 Genes and Proteins Rebecca S. LaRue, Valgerdur Andresdottir, Yannick Blanchard, Silvestro G. Conticello, David Derse, Michael Emerman, Warner C. Greene, Stefan R. Jonsson, Nathaniel R. Landau, Martin Lochelt, Harmit S. Malik, Michael H. Malim, Carsten Munk, Stephen J. O’Brien, Vinay K. Pathak, Klaus Strebel, Simon Wain-Hobson, Xiao-Fang Yu, Naoya Yuhki, and Reuben S. Harris*

The issue of ownership of digital watermark is addressed in this paper. An audio watermarking scheme is presented where the biometric pattern of iris is used to generate the watermark that has a stamp of ownership. The watermark is embedded in the high-energy regions selectively, which makes the embedding process robust against cropping and synchronization attacks. Experimental results of listening evaluation and robustness tests have confirmed that the proposed scheme maintains good perceptual property and offers strong resistance to typical signal processing attacks.

This paper proposes a method to separate audio signals when number of sources is more than the number of the sensors used and the number of source signals is not known in advance. The proposed method is for underdetermined blind source separation where time frequency masking is used to remove some of the signals from the mixtures till the number of mixtures is equal to the number of sources and then kurtosis maximization method is used to separate the remaining signals. In this paper we separate four signals from two mixtures under instantaneous conditions. The results obtained are better than conventional methods.

This paper proposes a method of generating pseudorandom number sequences based on biometric templates of iris image. These sequences are found to be unique in nature. Such sequences can be stored in a database for distinct identification of the extracted keys and they can act as secret keys for audio watermarking with a stamp of ownership unlike arbitrary pseudorandom number sequences and chaotic sequences. Correlation scores achieved under signal processing attacks is more than 0.9 that is significant for identification.

The reverse transcriptase enzyme plays an essential role in the HIV-1 life cycle by converting a single-stranded viral RNA genome into a double-stranded viral DNA through a complex process known as reverse transcription. The resulting double-stranded DNA is integrated into the host chromosome to form a provirus. A small proportion of the viral DNAs form dead-end circular products, which nevertheless can serve as useful surrogate markers for monitoring viral replication. Utilizing real-time PCR technology, it is possible to track and quantify different stages of the reverse transcription process, the proviruses, and the nonintegrated dead-end reverse transcription products.

Coinfection of two distinct retroviruses in the same host occurs in nature. For example, a significant human population is currently coinfected with human immunodeficiency virus type 1 (HIV-1) and HIV-2, two distinct lentiviruses (2, 19, 25). Phylogenetic analyses of the primate lentiviruses indicate that some of the viruses are recombinants of two distinct parental viruses (3, 37), indicating that the two parental viruses must coinfect the same host and infect the same target cells. Coinfection allows interactions between two viruses by several mechanisms including recombination and pseudotyping. Recombination generates a progeny different from the two parents; the best known example is that simian immunodeficiency virus SIVcpz, the zoonotic precursor of HIV-1, is thought to be a recombinant of SIVrcm and SIVgsn (3). Pseudotyping refers to virions containing components from two different viruses. A commonly observed example of pseudotyping is the use of Env proteins from different viruses, although pseudotyping can occur with many other virion components. Pseudotyped viruses often have properties different from those of virions containing components from one virus; for example, Env-pseudotyped viruses can have a different host range (27, 40). In all retroviruses, the major structural proteins are encoded by the gag gene, which is translated into the Gag polyprotein. During virus assembly, Gag coordinates the incorporation of other viral proteins and viral RNA and interacts with the host cell machinery to facilitate the release of viral particles (15, 20, 43). In orthoretroviruses, most of the newly released particles are “immature”; virus-encoded protease cleaves the Gag polyprotein during or soon after assembly to allow the transformation from immature to mature particles (1). This process, termed maturation, is required for the production of infectious virions. Although Gag polyproteins from different retroviruses might have only limited sequence homology, they share three similarly organized, conserved domains: matrix (MA), capsid (CA), and nucleocapsid (NC). Additionally, Gag from different retroviruses can have other domains. For example, the proteolytic cleavage of HIV-1 Gag also yields spacer peptide 1 (SP1 or p2), SP2 (p1), and p6 proteins. In the immature particles, Gag forms an approximately spherical shell underlying the membrane (51). After cleavage from the polyprotein, the HIV-1 CA (hCA) protein undergoes structural refolding and reassembles into a cone-shaped core that encloses the genomic RNA, NC, reverse transcriptase, integrase, and other viral and host components (1, 6, 8). It has been estimated that, although each immature HIV-1 particle contains around 5,000 Gag proteins, the mature shell of HIV-1 contains 1,000 to 1,500 CA proteins assembled into a mostly hexameric lattice (7). Therefore, only a fraction of CA proteins generated from Gag cleavage are used to form one mature virion core. We previously demonstrated that Gag proteins from HIV-1 and HIV-2 can coassemble and complement each other's functions (5). Because only a fraction of the mature CA proteins are used to generate a core, it is possible that only the CA proteins from one of the viruses are used to form a core. Coassembly of heterologous CA proteins into a core has not previously been demonstrated; therefore, it is unclear whether such a core can be formed and whether it is capable of conducting all steps leading to a successful infection. The mature CA proteins in the virion core play important roles in the viral replication cycle (14, 44, 45, 50, 52). After entry into the cells, virus uncoating occurs; CA proteins play an important role in regulating the uncoating process. Mutations in CA can lead to acceleration or delay of the uncoating events (14); both alterations can abolish the infectivity of HIV-1. Additionally, CA can influence the ability of HIV-1 to infect nondividing cells, possibly by affecting nuclear import (12, 53). Taken together, the identities of the CA proteins can affect the biological properties of the virus, and it is possible that a coassembled core can have properties different from those of the two parental viruses. We sought to determine whether CA proteins from two different primate lentiviruses can coassemble into a mature core to carry out all the steps necessary for infection. We envisioned three possibilities for virions with heterologous Gag proteins: first, mature heterologous CA proteins do not coassemble and the cores consist of CA proteins from one virus (pure CA cores); second, heterologous CA proteins coassemble into a core (mixed CA core) but viruses containing mixed cores are not infectious; and third, heterologous CA proteins coassemble into a mixed core and viruses containing mixed cores are infectious. To answer our experimental question, we exploited the inhibitory specificity of the tripartite motif 5α protein from the rhesus monkey (rhTRIM5α) for cores containing hCA but not SIVmac CA (sCA). TRIM5α is a member of the tripartite motif-containing family of proteins (35). The tripartite motif comprises a RING domain that includes two zinc finger motifs, one or two B-box domains, and a coiled-coil domain that mediates protein-protein interactions between TRIM family members (32, 41, 47). The rhTRIM5α protein restricts the replication of HIV-1 and HIV-2, but not SIVmac, a virus closely related to HIV-2 (22, 41, 54, 55). Although the mechanism by which rhTRIM5α restricts HIV-1 replication is not fully elucidated, recent studies suggest that rhTRIM5α targets the incoming mature HIV-1 core to promote premature uncoating and possibly degradation of CA proteins (9, 42). However, the inhibition of HIV-1 infection imposed by TRIM5α can be saturated by overwhelming the system with restriction-sensitive mature viruses or virus-like particles (21, 39, 41, 54). Interestingly, TRIM5α restriction cannot be saturated by adding immature virions or particles with cores consisting of restriction-insensitive CA proteins (10, 13). Furthermore, saturation of TRIM5α restriction depends on the stability of the incoming HIV-1 capsid (39). These results strongly support the idea that TRIM5α recognizes CA that has undergone conformational changes or the tertiary structure of CA in the mature core. N- and B-tropic murine leukemia viruses, two highly homologous virus strains that differ in their sensitivities to Fv-1 and TRIM5α, were used to demonstrate that coassembled viruses were sensitive to host Fv-1 restriction (26, 34, 46). The restriction imposed by rhTRIM5α is specific to hCA and is relieved in HIV-1 particles that have cores containing sCA proteins (33). We hypothesize that, if hCA and sCA can coassemble into the same core, TRIM5α can recognize hCA present in the mixed CA core and restrict the infection of such viruses; in contrast, a population of pure CA core viruses, some with sCA cores and some with hCA cores, would generate a different restriction pattern. To determine whether hCA and sCA can coassemble to form a mixed core, we examined the TRIM5α restriction phenotypes of viruses containing two different types of Gag proteins, one with hCA and the other with sCA. To ensure that most of the infection events observed were from coassembled viruses, we used two modified viruses that each harbored a debilitating gag mutation so that viruses derived from neither mutant could replicate efficiently. However, Gag proteins from two mutants could coassemble to allow functional complementation, thereby rescuing efficient virus replication. Our results indicate that hCA and sCA can coassemble into a mixed CA core to produce infectious virus.

During human immunodeficiency virus type 1 (HIV-1) assembly, the nucleocapsid (NC) and the PTAP motif in p6 of Gag play important roles in RNA encapsidation and virus release, respectively. We have previously demonstrated that functional complementation occurs between an NC mutant and a PTAP mutant to rescue viral replication. In this report, we examined the amounts of functional NC and PTAP motif that are required during virus replication. When NC and PTAP mutants were coexpressed at 5:1, 5:5, and 1:5 ratios, virus titers were rescued at 5%, 51%, and 86% of the wild-type level, respectively. These results indicate that HIV-1 requires a small amount of functional PTAP motif but far more functional NC to complete efficient replication. Further analyses reveal that RNA packaging can be significantly rescued in viruses containing a small amount of functional NC. However, most of the NC proteins must be functional to generate the wild-type level of R-U5 DNA product. Once the R-U5 product is generated, viruses containing half of the functional NC can complete reverse transcription and DNA integration at near-wild-type efficiency. These results define the quantitative requirements of NC and p6 during HIV-1 replication and provide insights into the requirement for the development of anti-HIV strategies using NC and p6 as targets.

In the preceding paper we described an experiment that determined the in vivo forward mutation rate in a single replication cycle for spleen necrosis virus. In addition to substitutions, frameshifts, and hypermutations, the mutated proviruses contained two classes of deletions. One class of deletions contained short direct repeats at the deletion junctions. Another class of deletions had short stretches of sequences inserted at the deletion junctions. In this report, we describe the deletion mutations, and we present models for their generation. Detailed analysis of two deletions with insertions indicates that these mutations occurred as a result of template switching during plus-strand DNA synthesis. The analysis also indicates that fragments of viral RNA generated by the viral RNase H endonuclease are used as templates and contribute to the sequences inserted at the deletion junctions.

Ad hoc networks are frequently established in insecure environments and hence become susceptible to attacks. A number of recently proposed secure routing protocols entail a number of prerequisites during both the network establishment and operation phases as they employ cryptographic algorithms. In contrast, trust-based routing protocols locate trusted routes and permit rapid deployment along with a dynamically adaptive operation, which conforms with the current network situation. In this paper, we evaluate the performance of our proposed Fuzzy based Trusted AODV routing protocol in a network, with varying number of malicious nodes. With the help of simulations, we demonstrate that the performance of the proposed protocol is better than AODV in terms of routing overhead ratio, throughput, latency and packet loss under similar attack conditions.

This paper presents an efficient surface inspection algorithm for cleavage site detection in broken 3D objects. Presented algorithm is intended to be used as a preprocessing stage for broken object reassembly. It helps reduce the area to be matched drastically by marking only the broken part of the surface resulting in much faster matches. The algorithm captures differences in the geometric structure in different parts of the 3D object represented by polygon meshes. These differences are used to find most probable cleavage/fracture sites. The presented approach is robust and the computations are simple making it time efficient, easy to implement and applicable to all triangulated surface data even in presence of noise. The algorithm finds extensive use in the field of computer vision, archeology, and computer aided orthopedic surgery etc. Index Terms-- Cleavage site detection, Roughness Estimation, 3D meshes.

We recently observed that mutations in the human immunodeficiency type 1 (HIV-1) reverse transcriptase (RT) connection domain significantly increase 3′-azido-3′-deoxythymidine (AZT) resistance up to 536 times over wild-type (WT) RT in the presence of thymidine analog resistance mutations (TAMs). These mutations also decreased RT template switching, suggesting that they altered the balance between nucleotide excision and template RNA degradation, which in turn increased AZT resistance. Several residues in the HIV-1 connection domain contact the primer strand and form an RNase H primer grip structure that helps to position the primer-template at the RNase H and polymerase active sites. To test the hypothesis that connection domain mutations enhanced AZT resistance by influencing the RNase H primer grip, we determined the effects of alanine substitutions in RNase H primer grip residues on nucleoside RT inhibitor resistance in the context of a WT, TAM-containing, or K65R-containing polymerase domain. Ten of the 11 RNase H primer grip mutations increased AZT resistance 20 to 243 times above WT levels in the context of a TAM-containing polymerase domain. Furthermore, all mutations in the RNase H primer grip decreased template switching, suggesting that they reduced RNase H activity. These results demonstrate that mutations in the RNase H primer grip region can significantly enhance AZT resistance and support the hypothesis that mutations in the connection and RNase H domains can increase resistance by altering the RNase H primer grip region, changing interactions between RT and the template-primer complex and/or shifting the balance between the polymerase and RNase H activities.

Replication of human immunodeficiency virus type 1 (HIV-1), like all organisms, involves synthesis of a minus-strand and a plus-strand of nucleic acid. Currently available PCR methods cannot distinguish between the two strands of nucleic acids. To carry out detailed analysis of HIV-1 reverse transcription from infected cells, we have developed a novel strand-specific amplification (SSA) assay using single-stranded padlock probes that are specifically hybridized to a target strand, ligated, and quantified for sensitive analysis of the kinetics of HIV-1 reverse transcription in cells. Using SSA, we have determined for the first time the ex vivo rates of HIV-1 minus-strand DNA synthesis in 293T and human primary CD4 + T cells (∼68 to 70 nucleotides/min). We also determined the rates of minus-strand DNA transfer (∼4 min), plus-strand DNA transfer (∼26 min), and initiation of plus-strand DNA synthesis (∼9 min) in 293T cells. Additionally, our results indicate that plus-strand DNA synthesis is initiated at multiple sites and that several reverse transcriptase inhibitors influence the kinetics of minus-strand DNA synthesis differently, providing insights into their mechanism of inhibition. The SSA technology provides a novel approach to analyzing DNA replication processes and should facilitate the development of new antiretroviral drugs that target specific steps in HIV-1 reverse transcription.

This paper presents algorithm for development of structural and continuous curved surface into a planar and non planar (radial) shape in 3D space. The development process is modeled by application of strain in certain plane from the curved surface to its planar development. A doubly curved surface has been generated for the purpose of technical studies. Important features of the approach include formulations of the coefficients of first fundamental form, second fundamental form, Gaussian curvature and Serret Frenet curve. The approximate strain field is obtained by solving a constrained linear and nonlinear problem in algorithm.