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a. In a single sentence (but in your own words, please), summarize the point of this figure.

Figure 1 shows that the reverse transcript of mutant virus cPPT-D is a linear stretch of double-stranded DNA that lacks the central DNA flap.

b. Diagram the synthesis of the genomic-length + strand DNA made from the cPPT-D mutant virus. Your diagram should include the site of initiation of this strand as well as any "jumps" or strand displacements that would be necessary.

2. (2 pts) Figure 2

a. The legend to part A states that "viral production was followed over time by RT activity in cell supernatants." Explain what is meant by the term "viral production," and why this is a measure of it.

In figure 2A, reverse transcription activity in PBL and MT4 cells is used as a measure of viral production. The experiment began by exposing each culture to equivalent amounts of viral particles. After exposure, excess viral components were removed. Samples of cell supernatant were then extracted and measured for presence of reverse transcriptase [activity]. Reverse transcriptase presence was used as an indication that the virus had inserted itself into its host genome, been subsequently transcribed and translated, [and had assembled and budded off from the host cell.]

b. Figure 2B is based on a measurement of beta-galactosidase activity. How is beta-gal being expressed in these cells, and what is this measurement designed to reflect? (You may need to consult the "Experimental procedures" section to answer this question.)

Beta-galactosidase is being expressed in these cells [from an integrated DNA construct] containing the lac Z gene under [the control of] an HIV-1 LTR. The HIV-1 LTR is responsible for transcription initiation and has recognition sites for HIV-1 genome transcriptional regulators. The protein tat is one of these regulators responsible for transcription of the entire HIV provirus. In this case, tat is reponsible for completing transcription of beta-gal on the [integrated] DNA construct. There is a catch, however: tat is encoded in HIV DNA. So, in order for tat to be produced, the HIV provirus must be integrated in the host cell genome, relying on partial transcription of the provirus to randomly produce tat which, in turn, initiates positive feedback on transcription levels; the more tat produced, the more transcription of the HIV provirus and its subsequent translation, producing more viral particles. Therefore, tat production signifies infection of a cell as well as successful [production, nuclear import and] integration of HIV proviral DNA. This figure shows the relative amount of infectivity of the wild-type and mutant strains of HIV by passively quantifying the amount of tat produced, a direct measure of successful infection of the host cell by HIV proviral DNA.

3. (2 pts) The data in figure 2 demonstrate that the cPPT-D mutant is deficient in the viral life cycle overall. Figure 3 presents experiments designed to assess particular stages of the HIV-1 life cycle. These experiments (and those in figures 4 and 5) are designed to determine the particular step at which the cPPT-D mutant is deficient.

At first glance, the experiment presented in figure 3A seems to parallel figure 2A. Both measure viral production. Yet the cPPT-D mutant is deficient in viral production as measured by the experiment in figure 2A, and proficient at it in the fig 3A experiment. Explain. (Hint: what steps of the viral life cycle are required for viral production in fig 2A, but not required in fig 3A? Why?)

Figure 2 assesses viral production by cPPT mutants, [which requires the entire HIV-1 life cycle.] Figure 3 assesses viral production by cPPT mutants after the mutant DNA has entered the nucleus, thereby concentrating the findings on late stages in the life cycle. Because the mutants were able to give rise to viral products only when they were already in the nucleus, it was concluded that the step which hinders the ability of mutants to produce viral products must come before the late stages. The significant step must be before the viral DNA enters the nucleus (or during!).

NOTE: Figure 2A requires attachment, entry, reverse transcription, nuclear import, and integration of the HIV provirus, none of which are required in the experiment presented in figure 3A. (Both experiments require HIV-1 gene expression, assembly, and budding.) Proficiency in the fig 3A experiment, but not in the fig 2A experiment, thus suggests that the defect of the cPPT-D mutant lies in attachment, entry, RT, nuclear import, or integration. Be careful not to overinterpret the data! These data do not show that there is a nuclear import defect--that comes later.

4. (2 pts) The "1 LTR" and "2 LTR" circular molecules diagrammed in figure 4A are commonly observed in HIV-1-infected cells. They are only found in the nuclei of these cells, not in the cytoplasm. They appear to be "by-products" of HIV-1 infection, not directly involved in the life cycle. Neither type of circle appears to be competent for integration into the host cell genome.

What cellular mechanisms do you know about that could explain the origin of these molecules? Explain. Your explanation should include why these molecules are only found in the nucleus. (Hint: you may need to use different cellular mechanisms to explain the origin of the 1-LTR and the 2-LTR circles.)

The circular viral DNA comes in two flavors: 1LTR and 2LTR. The 2LTR circular DNA could be a result of DNA repair mechanisms [i.e. non-homologous end-joining] acting in vivo. The retrotranscribed viral DNA is a linear molecule with blunt ends. These ends act as target sites for Ku-kinase repair machinery. The Ku/kinase complex binds to blunt-ended DNA and unwinds the helix, [exposing regions of] microhomology. [Ku doesn't create microhomologies, it just exposes them using its helicase activity.] These regions of microhomology bind via Watson-Crick base pairing. These DNA molecules are bound and act as a target for DNA ligase to ligate the sugar-phosphate backbones. If this happened to the viral DNA there would be 2 LTR sequences present on a now circular DNA molecule...

The 1LTR circles could be a result of homologous recombination. Since there are two LTR's of similar [identical] sequence and both are [oriented in the same direction] a simple loop could create alignment for homologous recombination. The spliced product would give rise to a single LTR within the circular DNA and a small double-stranded LTR sequence. This smaller sequence would most likely be degraded...These loops are limited to the nucleus due to the mucleus-specific location of the enzymes required.

NOTE that I asked for cellular, not viral mechanisms. (I specified this because formation of these circles does not depend on integrase protein.)

5. (1 pt) Figure 4C includes a quantitation of "integrated" virus. Explain how these numbers were determined. (You need not explain the entire assay in detail--just explain how these particular numbers were determined, and the reasoning behind that determination.)

In assay F4.B, total DNA, 2LTR DNA, 1LTR DNA, and linear DNA levels are quantified. The levels seen in F4.C are directly taken from the data in F4.B except the integrated DNA level. This level is determined by subtracting 2LTR DNA, 1LTR DNA, and linear DNA from the total amount of DNA. This assay assumes that these 4 categories that the viral DNA has been placed in account for all viral DNA.

6. (1 pt) What is the key finding of this paper, and why is it important?

The key finding of this paper is that the central DNA flap acts as a cis determinant of HIV-1 DNA [nuclear import]. Without this plus strand overlap, the viral genome couldn't enter the nucleus. [Since] the macrophages infected in primary [infection] are terminally differentiated, this function is essential to the propagation of the HIV-1 virus in humans. By determining the essential steps in the life cycle of the virus, we are given targets for possible sites of intervention.

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