Inhibitors of strand transfer that prevent integration and inhibit HIV-1 replication in cells.

Integrase is essential for human immunodeficiency virus-type 1 (HIV-1) replication; however, potent inhibition of the isolated enzyme in biochemical assays has not readily translated into antiviral activity in a manner consistent with inhibition of integration. In this report, we describe diketo acid inhibitors of HIV-1 integrase that manifest antiviral activity as a consequence of their effect on integration. The antiviral activity of these compounds is due exclusively to inhibition of one of the two catalytic functions of integrase, strand transfer.

Analysis of the intracellular transport properties of recombinant La Crosse virus glycoproteins.

The G1 and G2 glycoproteins of La Crosse virus, a member of the Bunyavirus genus of the Bunyaviridae, are encoded as a single open reading frame (ORF) in the viral middle-sized RNA segment. The primary product from this ORF is processed, either cotranslationally or shortly after translation, into the two glycoproteins and a nonstructural protein, NSm, of unknown function. We have expressed La Crosse glycoproteins using vaccinia vectors and studied their processing and localization. When expressed in the native G2-NSm-G1 configuration, both G1 and G2 targeted to the Golgi apparatus as shown by their colocalization with wheat germ agglutinin and acquired resistance to endoglycosidase H. When expressed independently, G2 was targeted to the Golgi apparatus but G1 was retained in the endoplasmic reticulum, indicating that a G1-G2 association is required for Golgi targeting of G1. In contrast to results with other members of the Bunyaviridae, we found that expression of G1 and G2 from separate vectors did not lead to the transport of the G1-G2 complex to the Golgi. However, disruption of the NSm region with a foreign sequence did not interfere with transport of the complex. When a portion of the beta-galactosidase gene was inserted in frame into NSm, the glycoproteins derived from this construct were processed and targeted properly and were capable of mediating cell-to-cell fusion.

Protection from La Crosse virus encephalitis with recombinant glycoproteins: role of neutralizing anti-G1 antibodies.

La Crosse virus, a member of the California serogroup of bunyaviruses, is an important cause of pediatric encephalitis in the midwestern United States. Like all bunyaviruses, La Crosse virus contains two glycoproteins, G1 and G2, the larger of which, G1, is the target of neutralizing antibodies. To develop an understanding of the role of each of the glycoproteins in the generation of a protective immune response, we immunized 1-week-old mice with three different preparations: a vaccinia virus recombinant (VV.ORF) that expresses both G1 and G2, a vaccinia virus recombinant (VV.G1) that expresses G1 only, and a truncated soluble G1 (sG1) protein prepared in a baculovirus system. Whereas VV.ORF generated a protective response that was mostly directed against G1, VV.G1 was only partially effective at inducing a neutralizing response and at protecting mice from a potentially lethal challenge with La Crosse virus. Nevertheless, a single immunization with the sG1 preparation resulted in a robust immune response and protection against La Crosse virus. These results indicate that (i) the G1 protein by itself can induce an immune response sufficient for protection from a lethal challenge with La Crosse virus, (ii) a neutralizing humoral response correlates with protection, and (iii) the context in which G1 is presented affects its immunogenicity. The key step in the defense against central nervous system infection appeared to be interruption of a transient viremia that occurred just after La Crosse virus inoculation.

Replication in cultured C2C12 muscle cells correlates with the neuroinvasiveness of California serogroup bunyaviruses.

The neuroinvasiveness of California serogroup bunyaviruses is determined by the ability of the virus to replicate in striated muscle after peripheral inoculation of mice. Neuroinvasiveness was mapped to the medium (M) RNA segment of the virus, which encodes the viral glycoproteins, when reassortants were made between La Crosse/original virus, a neuroinvasive isolate, and Tahyna-181/57 virus, a nonneuroinvasive clone. We have tested the murine muscle cell line C2C12 as a surrogate for myotropism and have found that there is a slight, but reproducible difference in the replication of virus clones bearing the M RNA segment of La Crosse/original virus compared to clones bearing the M RNA segment of Tahyna-181/57 virus, as determined by viral titer, antigen expression, and plaque formation.

Polygenic control of neuroinvasiveness in California serogroup bunyaviruses.

The pathogenesis of the California serogroup bunyaviruses includes both extraneural and intraneural replicative phases that can be separated experimentally. The present study dissects the viral genetic determinants of extraneural replication. We have previously described two attenuated reassortant clones of California serogroup bunyaviruses which exhibit reduced neuroinvasiveness after subcutaneous inoculation into suckling mice. Clone B1-1a bears an attenuated middle RNA segment (neuroinvasiveness phenotype v alpha v), and clone B.5 bears an attenuated large RNA segment (neuroinvasiveness phenotype alpha vv). We prepared reassortant viruses between these two strains and found that the two attenuated gene segments acted independently and additively, since reassortants bearing two attenuated RNA segments were more attenuated than the parental clones. Reassortants bearing no attenuated RNA segments were much more neuroinvasive than either parental clone, indicating that a neuroinvasive strain can be derived from two attenuated clones. Pathogenesis studies demonstrated that after injection of 10(3) PFU, the attenuated reassortant clones did not replicate in peripheral tissue, failed to reach the brain, and did not cause disease. At a dose of 10(6) PFU, attenuated clones failed to replicate to a significant level in peripheral tissue and produced only a minimal passive plasma viremia during the first 24 h but nevertheless reached high titers in the brain and killed mice. Because of this result, we investigated the possibility that neuroinvasion occurs via retrograde axonal transport, by determining whether sciatic nerve sectioning could protect against virus infection after hind leg footpad inoculation. We found that nerve sectioning had no effect on lethality, ruling out this mode of entry and suggesting that passive viremia is likely to be sufficient for invasion of the central nervous system.