Experimental cross-infections of Haemonchus placei (Place, 1893) in sheep and cattle.

To examine effects on biology and morphology, Haemonchus placei infections were propagated in cattle or sheep and infective larvae were introduced into the same or opposite host. Ovine source larvae had a geometric mean (GM) prepatent period of 26.5 days regardless of host species, compared to 30.6 days for bovine source larvae regardless of host species. Similarly, ovine source H. placei had a higher GM percentage establishment versus bovine source larvae (9.6% versus 3.4%) regardless of host species. The patent daily egg count for ovine source versus bovine source H. placei was 109.1 versus 50.0 FEC regardless of host species. Mean spicule length for ovine source H. placei was 492.5 microm while bovine source measured 496.5 microm. Mean female tail length for ovine source H. placei was 586.5 and 589.5 microm for bovine source H. placei. Using synlophe morphology, all worms that were measured were identified as H. placei. Sixty-three percent of females examined for vulvar flap morphology had knob-like vulvar flaps while the remaining 37% had linguiform vulvar flaps.

Interactions of HIV-1 nef with the mu subunits of adaptor protein complexes 1, 2, and 3: role of the dileucine-based sorting motif.

HIV-1 Nef interacts with cellular adaptor protein (AP) complexes and their medium (mu) subunits. However, the role of the dileucine-based sorting motif within Nef in these interactions has been incompletely characterized. Here, yeast two-hybrid assays indicated that HIV-1 Nef interacted not only with the mu subunits of AP-1 and AP-2, but also with that of AP-3. The interactions with mu1 and mu3 were markedly stronger than the interaction with mu2. Leucine residues of the sorting motif were required for the interactions with mu3 and mu2 and contributed to the interaction with mu1. Confocal immunofluorescence microscopy indicated that Nef, AP-1, and AP-3 (but not AP-2) were concentrated in a juxtanuclear region near the cell center, potentially facilitating interaction between Nef and the mu1 and mu3 subunits. However, leucine residues of the sorting motif were not required for this subcellular localization of Nef. These data suggest that the dileucine motif, required for optimal viral replication, functions through interactions with a variety of AP complexes, including AP-3, potentially by recruiting adaptor complexes to subcellular locations specified by additional determinants in the Nef protein.

Analysis of the SH3-binding region of HIV-1 nef: partial functional defects introduced by mutations in the polyproline helix and the hydrophobic pocket.

An SH3-binding domain within the Nef protein of primate lentiviruses has been reported to be important to viral replication and infectivity and dispensable for CD4 downregulation, but its precise role remains unclear. This study investigates the effects of mutations in both the polyproline helix and in the hydrophobic pocket that constitute the SH3-binding domain of Nef. The data demonstrate that the well-studied mutation of the central prolines is only partially disruptive to viral infectivity and replication. The central prolines also make a subtle contribution to the efficiency of CD4 downregulation, detectable only using low levels of Nef expression. Mutation of a conserved arginine in the polyproline helix abrogated more completely Nef-mediated enhancement of viral infectivity; this mutation also adversely affected CD4 downregulation at low levels of Nef expression. Only the R77A mutation substantially impaired downregulation of class I MHC. However, mutation of the central prolines and of R77 yielded proteins that were expressed less efficiently than wild-type Nef. The R77A mutant was expressed most poorly, compatible with its defective phenotypes in all assays. Mutations of the hydrophobic pocket were minimally detrimental to both the virologic and the receptor modulatory functions of Nef. Taken together, this analysis suggests that mutations in the SH3-binding domain do not abrogate fully any Nef-associated phenotype in the absence of detrimental effects on protein expression. We suggest that mutations in this domain can introduce incomplete effects caused by subtle impairments to protein expression; these effects may appear selective under certain experimental conditions due to different sensitivities of the assays to the level of Nef expression.

The dileucine-based sorting motif in HIV-1 Nef is not required for down-regulation of class I MHC.

A dileucine-based protein sorting motif has recently been identified within the C-terminal, solvent-exposed loop of HIV-1 Nef and has been shown to be required for Nef-mediated down-regulation of CD4 and for optimal viral infectivity. Here, we report that mutation of the dileucine motif has no effect on Nef-mediated down-regulation of class I MHC heavy chain. Instead, deletion of an acidic domain just N-terminal of the polyproline helix of the SH3-binding domain significantly impairs this function. These data indicate that down-regulation of class I MHC and CD4 are mechanistically distinct processes. The data also suggest that protein interactions mediated by the acidic domain, rather than by the dileucine motif, may contribute to this function of Nef.

Cell-associated viral RNA expression during acute infection with HIV type 1.

The mechanism of decline in viremia following acute infection with HIV is unknown. To characterize this process virologically, the expression of viral RNAs was analyzed in samples of peripheral blood mononuclear cells (PBMCs) from a patient who experienced a 100-fold decline in plasma viremia over a 13-day period prior to the initiation of antiretroviral therapy. Cell-associated viral RNA declined in association with the decline in plasma virus. During the initial 7 days of observation, plasma viremia declined more than 10-fold with no change in the ratio of unspliced to multiply spliced mRNAs. The efficiency of viral gene expression did not decline during the study period and varied from 380 to 2800 unspliced RNA copies per productively infected cell. Together, these data indicate no change in the relative proportion of cells in late- and early-stage gene expression during the initial decline and provide evidence against shortening of the viral replication cycle by immune surveillance. However, the prevalence of productively infected cells declined markedly during the 13 days of observation, from 1 in 250 to 1 in 25,000 PBMCs. These data are compatible with depletion of available target cells during the initial decline in viremia. As the level of plasma virus stabilized after 8 days of observation, the ratio of unspliced to multiply spliced mRNAs rose; this rise was due to a relatively greater decline in multiply spliced mRNA. These data suggest the possible onset of a blockade to new infection events (for example, by neutralizing antibody or chemokines), causing an increase in the relative proportion of cells in late-stage gene expression. They may also be explained, however, by the persistence of cell-associated virions together with the near disappearance of productively infected cells from the circulation.

Human immunodeficiency virus replication and genotypic resistance in blood and lymph nodes after a year of potent antiretroviral therapy.

Potent antiretroviral therapy can reduce human immunodeficiency virus (HIV) in plasma to levels below the limit of detection for up to 2 years, but the extent to which viral replication is suppressed is unknown. To search for ongoing viral replication in 10 patients on combination antiretroviral therapy for up to 1 year, the emergence of genotypic drug resistance across different compartments was studied and correlated with plasma viral RNA levels. In addition, lymph node (LN) mononuclear cells were assayed for the presence of multiply spliced RNA. Population sequencing of HIV-1 pol was done on plasma RNA, peripheral blood mononuclear cell (PBMC) RNA, PBMC DNA, LN RNA, LN DNA, and RNA from virus isolated from PBMCs or LNs. A special effort was made to obtain sequences from patients with undetectable plasma RNA, emphasizing the rapidly emerging lamivudine-associated M184V mutation. Furthermore, concordance of drug resistance mutations across compartments was investigated. No evidence for viral replication was found in patients with plasma HIV RNA levels of <20 copies/ml. In contrast, evolving genotypic drug resistance or the presence of multiply spliced RNA provided evidence for low-level replication in subjects with plasma HIV RNA levels between 20 and 400 copies/ml. All patients failing therapy showed multiple drug resistance mutations in different compartments, and multiply spliced RNA was present upon examination. Concordance of nucleotide sequences from different tissue compartments obtained concurrently from individual patients was high: 98% in the protease and 94% in the reverse transcriptase regions. These findings argue that HIV replication differs significantly between patients on potent antiretroviral therapy with low but detectable viral loads and those with undetectable viral loads.

Production and characterization of high-titer stocks of rev-defective HIV-1.

High-titer stocks of rev-defective HIV-1 virions have been produced and characterized. To produce these stocks, CEM cells transduced with a murine retroviral vector that expresses Rev were used to support the growth of an HIV-1 genome containing four inactivating mutations in the rev gene. The resulting viral stocks were of high-titer when used to infect CEM cells that expressed Rev, but had no measurable titer when used to infect parental CEM cells; replication-competent virus was not detected. As expected, these rev-defective viruses established early gene expression in parental CEM cells as demonstrated by the accumulation of viral mRNAs and Nef protein. The expression of late genes was impaired as demonstrated by the inability of these viruses to direct the accumulation of singly spliced mRNA or p24 antigen. These defective viruses did not interfere with replication of the wild-type virus, although they were produced at low levels during coinfection experiments. This experimental system may be useful for the study of early events during the HIV-1 life cycle. In addition, this system represents a simple method of gene transfer into CD4-positive cells using replication-defective but transcriptionally active HIV-1.

Cell surface CD4 downregulation and resistance to superinfection induced by a defective provirus of HIV-1.

Proviral sequences encoding defective HIV-1 regulatory genes have been detected previously in infected individuals; however, the role of these defective genomes in pathogenesis is unclear. The hypothesis that such replication-defective genomes might induce downregulation of the cellular receptor for HIV-1 (CD4) was tested. CEM cells were stably transfected with a provirus that contains a mutation in the splice site immediately 5' of the rev coding sequence. This mutant expresses early HIV-1 mRNAs but is defective for replication. Cells expressing this defective provirus displayed markedly reduced surface CD4. This downregulation of CD4 was dependent on an intact nef gene and was sufficient to cause resistance to superinfection by wild-type virus. These findings indicate that Nef as expressed by replication-defective HIV-1 can downregulate CD4. This perturbation of the T lymphocyte cell membrane is a potential basis for pathogenicity of defective HIV-1.

Biological importance and cooperativity of HIV-1 regulatory gene splice acceptors.

The replication of HIV-1 mutants containing altered splice acceptor sequences was studied. The splice acceptor sites 5' of the essential tat and rev AUG codons were altered to eliminate specifically spliced species from the viral repertoire of mRNAs. All splice site mutants were attenuated or fully defective. Mutation of the tat splice acceptor (exon 4) caused loss of the mRNA species containing exon 4 and resulted in an attenuated but replication-competent phenotype. Mutation of the rev splice acceptor sites resulted in viral genomes that failed to propagate in vitro. Mutation of the more 5' of the two major rev acceptors (exon 4A) caused loss of the mRNA species containing exon 4A together with a compensatory increase in use of the more 3' of the rev acceptors (exon 4B). Mutation of the splice acceptor for exon 4B caused the unexpected loss of both exon 4A- and 4B-containing mRNAs. In addition to these effects on the rev splice acceptors, mutations at the 4A and 4B sites also resulted in decreased use of the tat splice site (exon 4) located 175 nucleotides upstream. These effects on utilization of the tat splice acceptor site may explain the requirement for tat to efficiently complement these mutants. The 4A mutant was complemented by tat but not by rev. The 4B mutant was complemented by rev but required both tat and rev for maximum complementation. These data suggested a cooperativity among these splice sites necessary for efficient viral replication. They also indicated that while viral replication persisted at low levels in the absence of splicing to the known site 5' of the tat AUG, failure to splice to at least one of the two major sites 5' of the rev AUG resulted in insufficient rev activity for replication competence.