Sequence analysis of the HIV-1 protease coding region of 18 HIV-1-infected patients prior to HAART and possible implications on HAART.

BACKGROUND: The majority of HIV-infected patients are treated with highly active antiretroviral therapy (HAART) consisting of a combination of inhibitors of the protease (PIs) and the reverse transcriptase (RTIs). Analysis of mutations within these enzymes which are associated with development of resistance to the applied inhibitors is of major clinical importance. In particular, pre-existing mutations in previously untreated individuals may adversely influence the efficacy of HAART. OBJECTIVES: The sequences of the protease coding regions of 18 HIV-1-infected patients were analysed prior to HAART. STUDY DESIGN: DNA was extracted from whole blood samples of HIV-1-infected treatment-naive patients. The protease coding region was amplified by nested PCR and sequenced directly. The resulting amino acid substitutions were analysed for known mutations associated with known resistance to PIs. RESULTS: In all 18 analysed individuals we found 1-10 amino acid substitutions per patient in their HIV-1 protease coding region. These mutations occurred altogether at 27 positions of the 99 amino acids of the protease coding region. Seven of these mutated positions are associated with described resistance to PIs. Altogether, 15 of the 18 patients (83%) carried at least one such resistance-conferring alteration in their protease coding region. All patients are currently followed up during their present therapy to detect possible resistance formation to the applied PIs. CONCLUSIONS: A large variety of pre-existing mutations associated with resistance to PIs was observed prior to their treatment. As none of the patients ever received HAART before and infection with resistant viral strains is very unlikely, these amino acid substitutions evidently reflect natural polymorphism of the HIV-1 protease coding region.

Infection of nonhuman primate cells by pig endogenous retrovirus.

The ongoing shortage of human donor organs for transplantation has catalyzed new interest in the application of pig organs (xenotransplantation). One of the biggest concerns about the transplantation of porcine grafts into humans is the transmission of pig endogenous retroviruses (PERV) to the recipients or even to other members of the community. Although nonhuman primate models are excellently suited to mimic clinical xenotransplantation settings, their value for risk assessment of PERV transmission at xenotransplantation is questionable since all of the primate cell lines tested so far have been found to be nonpermissive for PERV infection. Here we demonstrate that human, gorilla, and Papio hamadryas primary skin fibroblasts and also baboon B-cell lines are permissive for PERV infection. This suggests that a reevaluation of the suitability of the baboon model for risk assessment in xenotransplantation is critical at this point.

[Risk of infection causes by viruses in xenotransplantation]. / Infektionsrisiko durch Viren bei Xenotransplantation.

The risk of transferring exogenous pig viruses to man during organ xenotransplantation can be controlled by keeping the pig donors pathogen-free. A risk of mobilizing pig endogenous retroviruses and a "xenozoonosis"-infection in human organ recipients cannot be excluded according to recently reported virological cell culture experiments. The present state of research, however, does not allow an answer to the question of whether or not a disease might be caused by such an infection and whether such a virus might be contagious to third persons.

Expression of pig endogenous retrovirus by primary porcine endothelial cells and infection of human cells.

BACKGROUND: The risk of interspecies transmission of retroviruses during xenotransplantation is suggested by reports of pig endogenous retrovirus (PERV) released from porcine cell lines productively infecting human cell lines in vitro and of infectious PERV being released from pig peripheral blood mononuclear cells after mitogenic stimulation. Endothelial cells are the main interface between a xenograft and the recipient's leucocytes and tissues. METHODS: We have analysed pig primary aortic endothelial cells (PAEC) together with other transplantation-relevant porcine cells and tissues for expression of PERV mRNA. Release of virus particles by PAEC was monitored by reverse transcriptase (RT) activity in the medium of cultured PAEC. Infectivity for human cells was tested by co-cultivation of irradiated PAEC with the human embryonal kidney cell line HEK293 and looking for virus release from the human cells. FINDINGS: PAECs, hepatocytes, lung, and skin from a variety of pig strains and breeds expressed PERV mRNA. PAEC released infectious particles. Co-cultivation of PAEC and HEK293 led to productive infection of the human cells and expression of PERV types A and B. INTERPRETATION: Release of infectious virus from PAEC occurred without mitogenic stimulation, suggesting a serious risk of retrovirus transfer after xenotransplantation.

Cleavage of the complement system C3 component by HIV-1 proteinase.

The C3 factor of the complement system and its C3b fragment are cleaved in vitro by the proteinase of the human immunodeficiency virus, type 1 (HIV PR). The cleavage occurs in the alpha-chain of both substrates at multiple sites yielding a 100 kDa fragment of the C3 alpha-chain and multiple fragments of the C3b alpha-chain. The scissile bonds are: Ala86-Glu87, Leu310-Leu311, His641-Trp642 and Arg649-Ser650. The resulting fragments resemble the physiologically occurring inactive fragments of C3: C3c and C3d, suggesting a possible biological role of the HIV-proteinase in the complement inactivation process.

Retroviral proteases: structure, function and inhibition from a non-anticipated viral enzyme to the target of a most promising HIV therapy.

Retrovirally encoded proteases are responsible for the maturation of immature viral particles yielding mature, infectious virus. This is done by apparent (auto)-processing and self-activation of the protease (PR) from a larger viral gag-PR-(pol) protein (zymogen) precursor and subsequent processing of the viral reverse transcriptase (RT) and integrase (IN), and the gag protein precursor into mature gag proteins. Only the matured components are capable of forming capsids for intact, infectious viruses. Blocking this proteolytic process results in production of immature, non-infective virions. All retroviral proteases are aspartic-type proteases. Determination of the three-dimensional structure revealed retroviral proteases as small, nearly symmetric homodimers. This prompted de novo design of inhibitors for the HIV protease taking advantage of the unique symmetric structure of the active center, unparalleled by cellular proteases. The novel substances inhibit in vitro the HIV protease at nanomolar/subnanomolar concentrations and exhibit very low toxicity. They are inactive against human proteases such as renin or pepsin. The HIV protease inhibitors (PI) represent a promising alternative to the reverse transcriptase (RT) inhibitors (AZT, ddC, ddI) hitherto used with limited success for HIV chemotherapy. Clinical studies confirmed the low toxicity but revealed a pharmacological pattern typical for these hydrophobic compounds, such as low water solubility, poor oral bioavailibility, and short plasma half-life. Typical for antimicrobial agents, also a resistance phenomenon became evident. Latest clinical results show, however, promisingly that both problems might be overcome by application of the PI in combination with RT inhibitors (such as AZT, ddI or ddC) exerting a remarkable synergistic antiviral effect with lasting restoration of the CD4-T-cell level.

In vivo processing of Pr160gag-pol from human immunodeficiency virus type 1 (HIV) in acutely infected, cultured human T-lymphocytes.

The processing of the HIV-1 Pr160gag-pol precursor polyprotein was analyzed in freshly HIV-1-infected MT-4 cultured cells. Single intermediates of the processing cascade were characterized by immunoblotting using distinct antisera. A potent inhibitor of the HIV protease (PR), Ro 31-8959, was employed to block cleavage by the mature PR, thus allowing insights into initial stages of the gag-pol (auto)-catalytical processing. While most known gag-pol cleavages were blocked in the presence of the inhibitor, the cleavage site between the gag-NC and the pol-p6 domains was still cleaved even in presence of high amounts (1 microM) of inhibitor, leading to the accumulation of a novel 114-kDa polyprotein comprising p6-PR-RT-IN. In the absence of inhibitor no accumulation of p114 was observed. In inhibitor-treated, HIV-1-infected cells a p6-PR intermediate was also detected, indicating subsequent cleavage of the PR/RT scissile bond. These results demonstrate initial cleavage(s) of the gag-pol precursor hydrolyzed by a proteolytic activity different from the mature PR and indicate that p114 (p6-PR-RT-IN) and p6-PR intermediates could play an essential role in the PR activation process.

Resistance of HIV type 1 to proteinase inhibitor Ro 31-8959.

During replication of human immunodeficiency virus type 1 (HIV-1), proteolytic cleavage of Gag and Gag-Pol precursor proteins into different functional protein subunits is catalyzed by the viral proteinase, and this enzyme is the target of the antiviral proteinase inhibitor, Ro 31-8959. We investigated in vitro which HIV mutants with reduced sensitivity to Ro 31-8959 emerged during proteinase inhibition treatment; from three different HIV-1 strains, comparable progeny virus resistant to proteinase inhibitor were found, whereas the same experimental protocol detected no resistant HIV-2 mutants. Molecular analysis of the mutations underlying resistance revealed a multistep mechanism in which an amino acid exchange was common to all resistant isolates, and in all experiments preceded further exchanges at position 90 (leucine to methionine) and/or at position 54 (isoleucine to valine). For wild-type strains the 90% inhibitory concentrations of Ro 31-8959 were close to 20 nM, whereas HIV-1 mutants with all 3 amino acid exchanges had more than 50-fold increased 90% inhibitory concentrations (above 1000 nM). The primary event (Gly-48 to valine) occurs at the hinge of the flaps of the proteinase, thus hampering entry of the inhibitor to the active center and suggesting steric hindrance. Detailed knowledge of this stereotypic process could open inhibitor design, thus preventing conceivable escape of resistant virus on proteinase inhibitor action.

Human immunodeficiency virus type 1 proteinase is rapidly and efficiently inactivated in human plasma by alpha 2-macroglobulin.

Human plasma impairs the activity of the human immunodeficiency virus (HIV-1) proteinase to cleave the HIV-1 gag-polyprotein precursor. The inhibition is due to the entrapment of the proteinase by plasma alpha 2-macroglobulin (alpha 2M). In methylamine-treated plasma, where alpha 2M is inactivated, HIV proteinase is not blocked. The interaction of alpha 2M and HIV-1 proteinase resulting in covalent complexes of proteinase and alpha 2M was demonstrated by immunoblotting with antiserum either to alpha 2M or to the HIV proteinase. We suggest if HIV-1 proteinase would be released in vivo from infected patients' cells, alpha 2M entrapment may prevent or minimize a conceivable cleavage of extracellular matrix or plasma proteins by the HIV-1 enzyme.

Prevalence of human T-cell lymphotropic virus infections in Germany.

The extent of human T-cell lymphotropic retrovirus HTLV-I and HTLV-II infections in the general population in central Europe has not been investigated fully. Two hundred forty-eight thousand blood donors from southern Germany were examined serologically for antibodies to the human lymphotropic retroviruses HTLV-I and HTLV-II: 0.021% were confirmed positive and 0.056% were "indeterminate". A limited number of seropositives and "indeterminate" samples were analyzed by polymerase chain reaction (PCR): the seropositives were confirmed as positive and 43% of the "indeterminate" samples were PCR-positive. The range of 0.021% HTLV-positives in 248,000 donors, i.e. about two in 10,000 individuals, mirrors closely the published data for the United States.

The Sukhumi primate monkey model for viral lymphomogenesis: high incidence of lymphomas with presence of STLV-I and EBV-like virus.

T-cell leukemia virus-like proviral sequences (STLV-I) as well as EBV-like sequences were detected in PBLs and tissues of non-human primates (Papio hamadryas baboons, Green monkeys and Macaca arctoides; Sukhumi Primate Center/Georgia) by PCR. Surprisingly, two different types of STLV-I within Papio hamadryas baboons were found. One of its represents the baboon prototype STLV-I-Su described earlier, present in lymphomatous baboons from the "high-lymphoma stock", which shows about 83% homology to HTLV-I and 85% to STLV-I in the env and tax genes. The inter-individual variability within this subtype is very low (about 1% in the tax gene). The second subtype was mainly found in asymptomatic animals from the control colony and showed in the env gene 95% homology to HTLV-I, but only 82% to the prototype baboon sequence. The presence of two subtypes within the Sukhumi baboon population might be interesting in respect to the inoculation experiments with human leukemic blood and to possible interspecies transmissions. The nature of the Herpes Papio-virus was elucidated as EBV-like and the homology to the human EBV was > 90% in the polymerase gene. The homologies between different monkey species were between 92 and 96% and also here two subtypes within the baboons were detected. This is the first direct demonstration by sequencing that the Herpes Papio virus is closely related to EBV. For further studies of this animal model, rabbits were inoculated with cells originated from lymphomatous baboons and macaques. The rabbits developed generalized lymphomas lethal within 1-2 months. EBV-like and STLV-I-like sequences could be detected by PCR and sequencing showed 99-100% identity to the inoculum, indicating in fact the transmission from monkey to rabbit. These animal models seem to be very suitable for the elucidation of the pathogenesis of human HTLV-I associated T-cell leukemia/lymphoma and might be further on used for therapeutical and preventative studies.

Detection and characterization of T-cell leukemia virus-like proviral sequences in PBL and tissues of baboons by PCR.

The "high lymphoma-prone" baboon stock (Papio hamadryas) of the Sukhumi Primate Center colony is characterized by a high prevalence of antibodies to the STLV-I/HTLV-I type of retrovirus and a high manifestation of human ATL-type (adult T-cell leukemia/lymphoma) malignancies (Yakovleva et al., this symposium). This is in contrast to other primate colonies and wild monkeys, which have low seroprevalence and very few if any ATL-type T-cell malignancies. To characterize the type of T-cell lymphoma retrovirus involved in the Sukhumi disease, a PCR (polymerase chain reaction) DNA analysis of peripheral blood lymphocytes (PBL) and of various tissues of healthy "at-risk", or ill baboons was performed. Proviral STLV/HTLV sequences were detected in all monkeys with symptoms of T-cell malignancy and/or antibodies to STLV-I/HTLV-I. For precise identification and characterization of the Sukhumi T-cell lymphoma virus, parts of the virus genome were mapped and sequenced from PCR derived fragments. A 420 nucleotide fragment of the env (gp 46) gene (analysed from 3 different DNA's) revealed 16.2% nucleotide divergence to the Japanese strain of HTLV-I and 14.8% to the Japanese strain of STLV-I including one deletion of a triplet. On the level of amino acid (a.a) sequence this revealed an exchange of 6 a.a. to STLV-I (4.3%), but only of 4 a.a. to HTLV-I (2.8%). The analysis of 120 nucleotides of the tax sequence (identical in 6 different DNAs) resulted in 5% nucleotide divergence to the HTLV-I (2.4% on the a.a. level) and 10% (7.3% a.a.) to the STLV-1. These results indicate that the Sukhumi T-cell lymphoma virus is a representative of the T-cell leukemia/lymphoma virus family, apparently more closely related to HTLV-I than to STLV-I genome. Furthermore, the infected monkeys from Sukhumi develop at a high rate a T-cell malignancy not observed among other baboons carrying STLV.

A seroepidemiological survey of antibodies to HTLV-I/HTLV-II in selected population groups in Paraguay.

Between March 1987 and November 1989 a cross-sectional serological survey was conducted on 884 residents of Paraguay to obtain data on the prevalence of antibodies to human T-cell leukemia virus type I/II (HTLV-I/II). Sera from 8/884 individuals (0.9%) were positive, confirmed by Western blotting and radioimmunoprecipitation (RIPA). This study shows that HTLV-I/II is very rare (or absent) among the general (healthy) population (0/338) and ethnic Japanese (0/227) in Paraguay. However, it can be detected at a rate of 2-3% in prostitutes (4/178) and homosexuals (4/117), suggesting sexual transmission as an important route for spread of HTLV-I/II in Paraguay.

Fibronectin is a non-viral substrate for the HIV proteinase.

The retrovirus encoded proteinase (PR) is required for the proper maturation of viral particles into infectious virus. The PR had been considered highly substrate specific, cleaving exclusively the viral gag and gag-pol protein precursor. It has recently been reported, however, that cytoskeleton and other cellular filament proteins can be cleaved by the HIV-1 PR. Here we have evidence that a cell-associated protein, the fibronectin (A-chain), is also cleaved in vitro specifically by this PR. The possibility of a cytotoxic role of the PR is conceivable.

Inhibition of the retroviral HIV-proteinase impairs maturation to infectious human immunodeficiency virus (HIV).

Newly developed inhibitors block the aspartic-type retroviral proteinase of the human immunodeficiency virus (HIV) at nanomolar concentration. The viral proteinase is responsible for the processing of viral encoded proteins. Applied to HIV infected cell culture, these inhibitors exhibit antiviral effects. The detailed analysis of these antiviral effects demonstrated that the synthesis of viral particles is only minimally decreased while the rate of infectious HIV particles is substantially reduced. The lack of infectivity is due to a failure in particle maturation which again is caused by the inhibition of the viral proteinase.

[HTLV-I antibody screening of blood donors]. / HTLV-I-Antikörperscreening bei Blutspendern.

Routine screening of 344,266 blood donations of approximately 200,000 donors for anti-HTLV-I was done by enzyme immuno assay (EIA). Repeatedly reactive samples were confirmed by Western Blot (WB) and radio immuno precipitation assay (RIPA). The prevalence of anti-HTLV-I in Bavarian blood donors is 0.02-0.026%. The specificity of HTLV-I-EIA is 99.95%.

Analysis of non-infectious HIV particles produced in presence of HIV proteinase inhibitor.

Newly developed substrate analogue peptidomimetics are able to inhibit the human immunodeficiency virus, HIV-1 proteinase at nanomolar concentration. In HIV infected cell culture they exhibit antiviral activity. We have analyzed the non-infectious HIV particles produced in chronically HIV infected cell culture in presence of one of these inhibitors. The total production of virus particles was not substantially reduced in drug treated cultures, compared to non-inhibited control cultures, but the infectivity of these virus particles was reduced about 100 fold. The processing of gag and gag-pol protein precursor was inhibited; only borderline activity of reverse transcriptase (RT) could be detected in these particles and they contained nonprocessed gag precursor protein. Thin section electron microscopy of inhibitor-treated, HIV-infected cells revealed reduced viral cytopathogenicity and both inhibition of particle assembly and incomplete maturation of the particles formed. The HIV particles produced in the presence of the proteinase inhibitor were studded with envelope glycoprotein knobs and often comprised multiple budding regions, but were morphologically immature.

Recombinant proteins VP1 and VP3 of hepatitis A virus prime for neutralizing response.

Six overlapping genomic regions of capsid proteins VP1 and VP3 of hepatitis A virus (HAV) inserted into the expression vectors pBD or pUR respectively expressed beta-galactosidase-HAV fusion proteins. The recombinant proteins were poorly soluble so they were difficult to detect by human anti-HAV sera in radioimmunoassay, but the fusion proteins dissolved in sodium dodecyl sulfate reacted with human and rabbit anti-HAV-positive sera in immunoblots. Antisera against VP1 and VP3 recombinant proteins reacted with the respective structural proteins of HAV in immunoblots. Two recombinant proteins, one including the first 120 amino acids of the N-terminus of VP1 and the other containing all of VP1 except for the first 60 N-terminal amino acids, induced a transient neutralizing antibody response in rabbits. Antisera directed against other regions of VP1 and VP3 neither neutralized viral infectivity nor recognized native virus in a competitive radioimmunoassay. However, when immunized animals were challenged with a sub-immunogenic dose of HAV, all animals responded with stable virus-neutralizing antibodies.